Project Summary Therapeutic antibodies against tumor targets have become standard therapeutic approach in oncology and have significantly improved clinical outcomes in liquid and solid tumors. In addition, therapeutic antibodies against immune checkpoint receptors have shown a great therapeutic potential in oncology when administered alone and in combination. However, adverse events related to presence of specific targets in normal tissues, especially in therapies combining two or more therapeutic antibodies, can limit dosing and thus efficacy of therapies, as well as prevent potential targets from being tested in clinical trials. Thus, a technology restricting therapeutic action to tumor sites would have a major impact on clinical efficacy of antibody therapeutics. ScalmiBio proposes to develop universal platform for converting any therapeutic antibody into activatable prodrug. Our technology acts to minimize interaction of an antibody therapeutic with normal tissue, and maximizes its target binding capability within tumor. We achieve this by exploiting tumor's own deregulated feature, activated proteases. Our technology thus allows antibody therapeutics to be activated in tumors by specific tumor present proteases. The goals of this project are 1) develop a novel technology that turns any therapeutic antibody into prodrug activated by tumor specific proteases and 2) convert an existing anti-CTLA4 therapeutic antibody into anti-CTLA4 antibody prodrug. In this 1-year feasibility study we will focus on: First, optimizing our universal shield which blocks interaction with target in normal tissues to generate anti-CTLA4 antibody prodrug, a validated target of great therapeutic potential in immuno-oncology. Second, design, optimize, and experimentally validate protease substrates for tumor specific therapeutic antibody activation. Protease substrates will be optimized for maximal cleavage in tumor tissues and minimal cleavage in serum, using mass spectrometry assay. Our collaborator, Omix Technologies has innovated chromatographic separations and mass spectrometry based data acquisition to develop high-throughput proteomics platform. Their methodology enables analysis of highly powered sample sets in a time- and cost-efficient manner. Third, our technology and protease substrates will be validated in-vivo in mouse colon carcinoma syngeneic mouse model sensitive to anti-CTLA4 immunotherapy. ScalmiBio's technology platform will provide several benefits over typical antibody therapeutic: 1) minimize unwanted side-effects of antibody therapeutics 2) unlock potential of new targets 3) result in more predictable pharmacokinetics.